EXCEEDTHE SPACE PROVIDED. This MERIT application examines regulation of signaling by the substance P (SP) and its neurokinin 1 receptor (NK1R), which regulate inflammation, pain and gastrointestinal function. Completed studies determined the mechanisms of SP-induced trafficking of the NK1R, investigated the importance of trafficking to signal transduction, and determined the mechanism that terminate the proinflammatory actions of SP in intact animals. Studies in cell lines revealed that the NK1R interacts with arrestins and internalizes by dynamin-mediated mechanism. Internalized NK1R either recycles by rabSa, rab4a and rabtla-dependent mechanisms, or is ubiquitinated and sorted to lysosomes for degradation. NK1R phosphorylation and interaction with arrestins mediate desensitization, whereas NK1R endocytosis, dissociation from arrestins and SP, and recycling mediate resensitization. Arrestins also recruit src to the NK1R in endosomes, which is required for mitogenic signaling. Studies in intact animals revealed that the NK1R internalizes and desensitizes at sites of inflammation. Disruption of mechanisms that terminate SP signaling, by deleting the SP degrading enzyme neutral endopeptidase, exacerbated SP-mediated inflammation. Proposed experiments extend these accomplishments.
Aim 1 examines mechanisms of endocytic sorting of receptors to lysosomes or the plasma membrane. Experiments will examine the importance of NK1R ubiquitination to lysosomal trafficking, and will determine the role of the E3 ubiquitin ligase c-Cbl and the endocytic sorting proteins Hrs, TsglOO and Vsp4 in this process.
Aim 2 will investigate the role of arrestins in the formation and function of signaling modules containing mitogen activate protein kinases. By comparing receptors that interact with arrestins with different affinities, the importance of this mechanism for mitogenic signaling of different neurokinin receptors will be determined.
Aim 3 will determine the importance of arrestin-dependent endocytosis and desensitization of the NK1R in terminating the proinflammatory actions of SP in the intact animal. By studying arrestin knockout mice or by down-regulating arrestins using RNA interference, important new information will be obtained about the relevance of receptor desensitization in limiting the pathophysiological actions of SP. PERFORMANCE SITE ========================================Section End===========================================
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